Use of SLIM-1 in the assessment of heart failure

ABSTRACT

The invention relates to a method for assessing heart failure in vitro comprising the steps of measuring in a sample the concentration of the marker SLIM-1, of optionally measuring in the sample the concentration of one or more other marker(s) of heart failure, and of assessing heart failure by comparing the concentration determined in for SLIM-1 and the concentration(s) determined for the optionally one or more other marker to the concentration of this marker or these markers as established in a reference population. Also disclosed are the use of SLIM-1 as a marker protein in the assessment of heart failure, a marker combination comprising SLIM-1 and a kit for measuring SLIM-1.

RELATED APPLICATIONS

This application is a continuation of PCT/EP2008/001842 filed Mar. 7,2008 and claims priority to EP 07004740.2 filed Mar. 8, 2007.

FIELD OF THE INVENTION

The present invention relates to a method for assessing heart failure inan individual comprising the steps of a) measuring in a sample obtainedfrom the individual the concentration of the marker SLIM-1, of b)optionally measuring in the sample the concentration of one or moreother marker(s) of heart failure, and of assessing heart failure bycomparing the concentration determined in step (a) and optionally theconcentration(s) determined in step (b) to the concentration of thismarker or these markers as established in a control sample. Alsodisclosed are the use of SLIM-1 as a marker protein in the assessment ofheart failure, a marker combination comprising SLIM-1 and a kit formeasuring SLIM-1.

BACKGROUND OF THE INVENTION

Heart failure (HF) is a major and growing public health problem. In theUnited States for example approximately 5 million patients have HF andover 550 000 patients are diagnosed with HF for the first time each year(In: American Heart Association, Heart Disease and Stroke Statistics:2005 Update, Dallas, Tex.; American Heart Association (2005)). SimilarlyUS-statistics show that HF is the primary reason for 12 to 15 millionoffice visits and 6.5 million hospital days each year. From 1990 to1999, the annual number of hospitalizations has increased fromapproximately 810 000 to over 1 million for HF as a primary diagnosisand from 2.4 to 3.6 million for HF as a primary or secondary diagnosis.In 2001, nearly 53 000 patients died of HF as a primary cause. Heartfailure is primarily a condition of the elderly, and thus the widelyrecognized “aging of the population” also contributes to the increasingincidence of HF. The incidence of HF approaches 10 per 1000 in thepopulation after age 65. In the US alone, the total estimated direct andindirect costs for HF in 2005 were approximately $27.9 billion andapproximately $2.9 billion annually is spent on drugs for the treatmentof HF (cf. the above cited AHA-statistics).

Heart Failure

Heart Failure is characterized by a loss in the heart's ability to pumpas much blood as the body needs. Failure does not mean that the hearthas stopped pumping but that it is failing to pump blood as effectivelyas it should.

The NYHA (New York Heart Association) and the ACC/AHA (AmericanAssociation of Cardiology/American Heart Association) have bothestablished functional classes of HF to gauge the progression of thedisease. The NYHA classification scheme has four classes of diseasestate: Class 1 is asymptomatic at any level of exertion. Class 2 issymptomatic at heavy exertion and Classes III and IV are symptomatic atlight and no exertion, respectively.

In the four stage ACC/AHA scheme, Stage A is asymptomatic but is at riskfor developing HF. Stage B there is evidence of cardiac dysfunctionwithout symptoms. In Stage C there is evidence of cardiac dysfunctionwith symptoms. In Stage D, the subject has symptoms of HF despitemaximal therapy.

Etiology of HF

Medically, heart failure (HF) must be appreciated as being a complexdisease. It may be caused by the occurrence of a triggering event suchas a myocardial infarction (heart attack) or be secondary to othercauses such as hypertension, diabetes or cardiac malformations such asvalvular disease. Myocardial infarction or other causes of HF result inan initial decline in the pumping capacity of the heart, for example bydamaging the heart muscle. This decline in pumping capacity may not beimmediately noticeable, due to the activation of one or morecompensatory mechanisms. However, the progression of HF has been foundto be independent of the patient's hemodynamic status. Therefore, thedamaging changes caused by the disease are present and ongoing evenwhile the patient remains asymptomatic. In fact, the compensatorymechanisms which maintain normal cardiovascular function during theearly phases of HF may actually contribute to progression of the diseasein the long run, for example by exerting deleterious effects on theheart and its capacity to maintain a sufficient level of blood flow inthe circulation.

Some of the more important pathophysiological changes which occur in HFare (i) activation of the hypothalamic-pituitary-adrenal axis, (ii)systemic endothelial dysfunction and (iii) myocardial remodeling.

(i) Therapies specifically directed at counteracting the activation ofthe hypothalamic-pituitary-adrenal axis include beta-adrenergic blockingagents (B-blockers), angiotensin converting enzyme (ACE) inhibitors,certain calcium channel blockers, nitrates and endothelin-1 blockingagents. Calcium channel blockers and nitrates, while producing clinicalimprovement have not been clearly shown to prolong survival, whereasB-blockers and ACE inhibitors have been shown to significantly prolonglife, as have aldosterone antagonists. Experimental studies usingendothelin-1 blocking agents have shown a beneficial effect.

(ii) Systemic endothelial dysfunction is a well-recognized feature of HFand is clearly present by the time signs of left ventricular dysfunctionare present. Endothelial dysfunction is important with respect to theintimate relationship of the myocardial microcirculation with cardiacmyocytes. The evidence suggests that microvascular dysfunctioncontributes significantly to myocyte dysfunction and the morphologicalchanges which lead to progressive myocardial failure.

In terms of underlying pathophysiology, evidence suggests thatendothelial dysfunction may be caused by a relative lack of NO which canbe attributed to an increase in vascular O₂-formation by anNADH-dependent oxidase and subsequent excess scavenging of NO. Potentialcontributing factors to increased O₂-production include increasedsympathetic tone, norepinephrine, angiotensin endothelin-1 and TNF-α. Inaddition, levels of IL-10, a key anti-inflammatory cytokine, areinappropriately low in relation to TNF-α levels. It is now believed thatelevated levels of TNF-α, with associated proinflammatory cytokinesincluding IL-6, and soluble TNF-α receptors, play a significant role inthe evolution of HF by causing decreased myocardial contractility,biventricular dilatation, and hypotension and are probably involved inendothelial activation and dysfunction. It is also believed that TNF-αmay play a role in the hitherto unexplained muscular wasting whichoccurs in severe HF patients. Preliminary studies in small numbers ofpatients with soluble TNF-receptor therapy have indicated improvementsin NYHA functional classification and in patient well-being, as measuredby quality of life indices.

(iii) Myocardial remodeling is a complex process which accompanies thetransition from asymptomatic to symptomatic heart failure, and may bedescribed as a series of adaptive changes within the myocardium, likealterations in ventricular shape, mass and volume (Piano, M. R., et al.,J. Cardiovasc. Nurs. 14 (2000) 1-23, quiz 119-120; Molkentin, J. D.,Ann. Rev. Physiol. 63 (2001) 391-426). The main components of myocardialremodeling are alterations in myocyte biology, like myocyte hypertrophy,loss of myocytes by necrosis or apoptosis, alterations in theextracellular matrix and alterations in left ventricular chambergeometry. It is unclear whether myocardial remodeling is simply theend-organ response that occurs following years of exposure to the toxiceffects of long-term neurohormonal stimulation, or whether myocardialremodeling contributes independently to the progression of heartfailure. Evidence to date suggests that appropriate therapy can slow orhalt progression of myocardial remodeling.

Markers and Disease State

As indicated above, myocyte hypertrophy is likely to represent one ofthe first steps down the road to HF. Myocyte hypertrophy ischaracterized by an increased expression of some genes encodingcontractile proteins, such as p-myosin heavy chain and troponin T (TnT),and of some non-contractile proteins, such as A-type and B-typenatriuretic peptides, by an increased cell size and by cytoskeletalalteration (Piano, M. R., et al., J. Cardiovasc. Nurs. 14 (2000) 1-23,quiz 119-120; Molkentin, J. D., Ann. Rev. Physiol. 63 (2001) 391-426).

Studies of human and animal models of heart failure suggest depressedmyocyte function in the later stages of cardiac failure. The mechanismsthat underlie myocyte dysfunction have been suggested to involvealterations in the calcium-handling network, myofilament andcytoskeleton (de Tombe, P. P., Cardiovasc. Res. 37 (1998) 367-380). Forexample, in human and animal models of heart failure, sarcoplasmicreticulum calcium-ATPase enzyme activity is reduced, while both mRNA andprotein levels of the sarcolemmal Na+/Ca2+ exchanger are increased.Moreover, there is isoform-switching of TnT, reduced phosphorylation oftroponin I (TnI), decreased myofibrillar actomyosin ATPase activity andenhanced microtubule formation in both human, and animal models of heartfailure.

Initially the changes to the heart, leading to myocardial remodeling aremeant to compensate for the diseased parts of the myocardium in order tosustain the body's demand for oxygen and nutrients. However, thecompensatory phase of heart failure is limited, and, ultimately, thefailing heart is unable to maintain cardiac output adequate to meet thebody's needs. Thus, there is a transition from a compensatory phase to adecompensatory phase. In the decompensatory phase, the cascade ofchanges in the heart continues but is no longer beneficial, moving thepatient down the progression of heart failure to a chronic state andeventual death.

According to the “ACC/AHA 2005 Guideline Update for the Diagnosis andManagement of Chronic Heart Failure in the Adult”, the disease continuumin the area of heart failure is nowadays grouped into four stages asnoted above. In stages A and B the individuals at risk of developingheart failure are found, whereas stages C and D represent the groups ofpatients showing signs and symptoms of heart failure. Details fordefining the different stages A through D as given in the abovereference are hereby included by reference.

Diagnostic Methods in Heart Failure

The single most useful diagnostic test in the evaluation of patientswith HF is the comprehensive 2-dimensional echocardiogram coupled withDoppler flow studies to determine whether abnormalities of myocardium,heart valves, or pericardium are present and which chambers areinvolved. Three fundamental questions must be addressed: 1) is the LVEFpreserved or reduced, 2) is the structure of the LV normal or abnormal,and 3) are there other structural abnormalities such as valvular,pericardial, or right ventricular abnormalities that could account forthe clinical presentation? This information should be quantified with anumerical estimate of EF, measurement of ventricular dimensions and/orvolumes, measurement of wall thickness, and evaluation of chambergeometry and regional wall motion. Right ventricular size and systolicperformance should be assessed. Atrial size should also be determinedsemiquantitatively and left atrial dimensions and/or volumes measured.

Noninvasive hemodynamic data acquired at the time of echocardiographyare an important additional correlate for patients with preserved orreduced EF. Combined quantification of the mitral valve inflow pattern,pulmonary venous inflow pattern, and mitral annular velocity providesdata about characteristics of LV filling and left atrial pressure.Evaluation of the tricuspid valve regurgitant gradient coupled withmeasurement of inferior vena caval dimension and its response duringrespiration provides an estimate of systolic pulmonary artery pressureand central venous pressure.

Stroke volume may be determined with combined dimension measurement andpulsed Doppler in the LV outflow tract. However, abnormalities can bepresent in any of these parameters in the absence of HF. No one of thesenecessarily correlates specifically with HF; however, a totally normalfilling pattern argues against clinical HF.

From a clinical perspective, the disease is clinically asymptomatic inthe compensatory and early decompensatory phases (completelyasymptomatic in stage A and with structural heart disease but no signsand symptoms of HF in stage B, cf. the ACC/AHA practice guidelines).Outward signs of the disease (such as shortness of breath) do not appearuntil well into the decompensatory phase (i.e., stages C and D accordingto the ACC/AHA guidelines). Current diagnosis is based on the outwardsymptoms of patients in stages C and D.

Typically patients with heart failure receive a standard treatment withdrugs that interact with specific mechanisms involved in heart failure.There are no diagnostic tests that reflect those specific mechanismsreliably and help the physician to choose the right drug (and dose) forthe right patient (e.g., ACE inhibitor, AT II, β-blockers, etc).

Prior Diagnosis of HF with Markers

Early assessment of patients at risk for heart failure appears to bepossible only by biochemical markers since the individual at risk ofdeveloping heart failure at that stage is still free of clinical HFsymptoms. There are no established biochemical markers currentlyavailable for the reliable pre-symptomatic assessment of the disease. Bythe time the diagnosis HF is established nowadays, the disease isalready well underway.

The natriuretic peptide family, especially the atrial natriureticpeptide family and the brain natriuretic peptide family have in recentyears proven to be of significant value in the assessment of HF.

HF Prognosis and Need

At least partially due to the late diagnosis, 50% of patients with HFdie within two years of diagnosis. The 5-year survival rate is less than30%. There is a significant need for new biochemical markers aiding inthe early diagnosis of heart failure.

An improvement in the early assessment of individuals at risk for heartfailure, i.e., of individuals that are clinically asymptomatic for heartfailure is warranted.

It has been established in recent years that B-type natriuretic peptidemarkers represent an excellent tool to monitor disease progression inpatients with HF and to assess their risk of cardiovascularcomplications, like heart attack.

However, as for many other diagnostic areas a single marker is notsufficient.

Whereas a low value of NT-proBNP has a very high negative predictivevalue for ruling out HF or LVD, the positive predictive value for heartfailure in the above and other studies (cf. Triepels, R. H., et al.,Clin. Chem. 49, Suppl. (2003) A37-38) has been found to be in the rangeof 50-60%. Thus a marker useful in assessing individuals at risk forheart failure that on its own e.g., has a high, or in combination withNT-proBNP, and as compared to NT-proBNP alone has an improved positivepredictive value for HF is of high clinical/practical importance.

A marker aiding in the assessment of a patient with heart failure alsois of high importance to achieve further technical progress in thisclinically very important and demanding diagnostic area.

SUMMARY OF THE INVENTION

It has now been found and established that the marker SLIM-1 can aid inthe assessment of heart failure. In one embodiment it can help to assesswhether an individual is at risk of developing heart failure. In afurther aspect it can aid in the assessment of disease progression. Inanother embodiment it can aid in predicting the onset of heart failure.In another embodiment it can aid in assessing and selecting anappropriate treatment regime to prevent or treat heart failure.

Disclosed herein is a method for assessing heart failure in anindividual comprising the steps of measuring in a sample obtained fromthe individual the concentration of the marker SLIM-1, of optionallymeasuring in the sample the concentration of one or more other marker(s)of heart failure, and of assessing heart failure by comparing theconcentration of SLIM-1 and optionally the concentration(s) of the oneor more other marker to the concentration of this marker or thesemarkers as established in a control sample.

The invention also relates to the use of protein SLIM-1 as a markermolecule in the assessment of heart failure.

Further disclosed is the use of a marker combination comprising SLIM-1and one or more other marker of heart failure in the assessment of heartfailure.

Also provided is a kit for performing the method for assessing heartfailure in vitro comprising the steps of measuring in a sample theconcentration of the marker SLIM-1, of optionally measuring in thesample the concentration of one or more other marker(s) of heartfailure, and of assessing heart failure by comparing the concentrationof SLIM-1 and optionally the concentration(s) of the one or more othermarker to the concentration of this marker or these markers asestablished in a reference population, the kit comprising the reagentsrequired to specifically measure SLIM-1 and the optionally one or moreother marker of heart failure.

Additional aspects and advantages of the present invention will beapparent in view of the description which follows. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Phenotypic analyses of wildtype and R9C mice. (A) Survival curvesfor wildtype mice (n=79) and R9C mice (n=44) are generated following a24 week period. (B) Cardiac shortening assessed by echocardiography(=fractional shortening). Significant functional impairment in the R9Ctransgenic animals begin as early as 8 weeks of age.

FIG. 2 Echocardiographic and hemodynamic parameters in wildtype and ABmice. (A) Changes in maximum pressure in mmHg at 2, 4, and 8 weeks postsurgery. (B) Change in % left ventricular ejection fraction (LVEF) at 2,4, and 8 weeks after surgery. (Closed circles indicate the data fromsham operated mice and open circles indicate the data from mice withaortic binding (AB).

FIG. 3 Western Blotting data as obtained with cardiac tissue from R9Cand control mice, respectively. A strong overexpression of SLIM-1 isobserved in tissue samples derived from experimental (R9C) animalssuffering from heart failure versus tissue samples derived from healthymice (=+/+). Numbers underneath the stained bands indicate relativeexpression levels determined by the numbers of mass spectra recorded.

FIG. 4 SLIM-1 measured in 10 HF and control samples, respectively.Optical densities (ODs) in the SLIM-1 assay are given for samplesderived from patients with heart failure are labeled (HF=rhombi), andfor healthy controls (normal human serum=NHS=squares), respectively.

FIG. 5 SLIM-1 measured in 10 HF and control samples, respectively. ODsare given for SLIM-1 as measured in samples derived from patients withheart failure are labeled (HF) and in samples from healthy controls(normal human serum=NHS), respectively. The box-and-whisker-blots showthe lower and upper quartiles (boxes) as well as the highest and lowestvalues (whiskers).

DETAILED DESCRIPTION OF THE INVENTION

In a first embodiment the present invention relates to a method forassessing heart failure in an individual comprising the steps of a)measuring in a sample obtained from the individual the concentration ofthe marker SLIM-1, b) optionally measuring in the sample theconcentration of one or more other marker(s) of heart failure, and c)assessing heart failure by comparing the concentration determined instep (a) and optionally the concentration(s) determined in step (b) tothe concentration of this marker or these markers as established in acontrol sample.

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an antibody” means one antibody or morethan one antibody.

The expression “one or more” denotes 1 to 50, preferably 1 to 20 alsopreferred 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or 15.

The term “marker” or “biochemical marker” as used herein refers to amolecule to be used as a target for analyzing a patient's test sample.In one embodiment examples of such molecular targets are proteins orpolypeptides. Proteins or polypeptides used as a marker in the presentinvention are contemplated to include naturally occurring fragments ofsaid protein, in particular, immunologically detectable fragments.Immunologically detectable fragments preferably comprise at least 6, 7,8, 10, 12, 15 or 20 contiguous amino acids of said marker polypeptide.One of skill in the art would recognize that proteins which are releasedby cells or present in the extracellular matrix may be damaged, e.g.,during inflammation, and could become degraded or cleaved into suchfragments. Certain markers are synthesized in an inactive form, whichmay be subsequently activated by proteolysis. As the skilled artisanwill appreciate, proteins or fragments thereof may also be present aspart of a complex. Such complex also may be used as a marker in thesense of the present invention. In addition, or in the alternative, amarker polypeptide may carry a post-translational modification. Examplesof posttranslational modifications amongst others are glycosylation,acylation, and/or phosphorylation.

The term “assessing heart failure” is used to indicate that the methodaccording to the present invention will aid the physician to assesswhether an individual is at risk of developing heart failure, or aid thephysician in his assessing of an HF patient in one or several otherareas of diagnostic relevance in HF. Preferred areas of diagnosticrelevance in assessing an individual with HF are the staging of heartfailure, differential diagnosis of acute and chronic heart failure,judging the risk of disease progression, guidance for selecting anappropriate drug, monitoring of response to therapy, and the follow-upof HF patients.

A “marker of heart failure” in the sense of the present invention is anymarker that if combined with the marker SLIM-1 adds relevant informationin the assessment of HF to the diagnostic question under investigation.The information is considered relevant or of additive value if at agiven specificity the sensitivity, or if at a given sensitivity thespecificity, respectively, for the assessment of HF can be improved byincluding said marker into a marker combination already comprising themarker SLIM-1. Preferably the improvement in sensitivity or specificity,respectively, is statistically significant at a level of significance ofp=0.05, 0.02, 0.01 or lower. Preferably, the one or more other marker ofheart failure is selected from the group consisting of a natriureticpeptide marker, a cardiac troponin marker, and a marker of inflammation.

The term “sample” as used herein refers to a biological sample obtainedfor the purpose of evaluation in vitro. In the methods of the presentinvention, the sample or patient sample preferably may comprise any bodyfluid. Preferred test samples include blood, serum, plasma, urine,saliva, and synovial fluid. Preferred samples are whole blood, serum,plasma or synovial fluid, with plasma or serum representing the mostconvenient type of sample. As the skilled artisan will appreciate, anysuch assessment is made in vitro. The patient sample is discardedafterwards. The patient sample is solely used for the in vitro method ofthe invention and the material of the patient sample is not transferredback into the patient's body. Typically, the sample is a liquid sample,e.g., whole blood, serum, or plasma.

The expression “comparing the concentration . . . to the concentrationas established in a control sample” is merely used to further illustratewhat is obvious to the skilled artisan anyway. The control sample may bean internal or an external control sample. In one embodiment an internalcontrol sample is used, i.e. the marker level(s) is(are) assessed in thetest sample as well as in one or more other sample(s) taken from thesame subject to determine if there are any changes in the level(s) ofsaid marker(s). In another embodiment an external control sample isused. For an external control sample the presence or amount of a markerin a sample derived from the individual is compared to its presence oramount in an individual known to suffer from, or known to be at risk of,a given condition; or an individual known to be free of a givencondition, i.e., “normal individual”. For example, a marker level in apatient sample can be compared to a level known to be associated with aspecific course of disease in HF. Usually the sample's marker level isdirectly or indirectly correlated with a diagnosis and the marker levelis e.g. used to determine whether an individual is at risk for HF.Alternatively, the sample's marker level can e.g. be compared to amarker level known to be associated with a response to therapy in HFpatients, the differential diagnosis of acute and chronic heart failure,the guidance for selecting an appropriate drug to treat HF, in judgingthe risk of disease progression, or in the follow-up of HF patients.Depending on the intended diagnostic use an appropriate control sampleis chosen and a control or reference value for the marker establishedtherein. It will be appreciated by the skilled artisan that such controlsample in one embodiment is obtained from a reference population that isage-matched and free of confounding diseases. As also clear to theskilled artisan, the absolute marker values established in a controlsample will be dependent on the assay used. Preferably samples from 100well-characterized individuals from the appropriate reference populationare used to establish a control (reference) value. Also preferred thereference population may be chosen to consist of 20, 30, 50, 200, 500 or1000 individuals. Healthy individuals represent a preferred referencepopulation for establishing a control value. In one embodiment thecontrol sample will be an internal control sample. In this embodimentserial samples are obtained from the individual under investigation andthe marker levels are compared.

An increased value for SLIM-1 as measured from a sample derived from anindividual is indicative for heart failure.

The values for SLIM-1 as measured in a control group or a controlpopulation are for example used to establish a cut-off value or areference range. A value above such cut-off value or out-side thereference range and its higher end is considered as elevated.

In a one embodiment a fixed cut-off value is established. Such cut-offvalue is chosen to match the diagnostic question of interest.

In one embodiment values for SLIM-1 as measured in a control group or acontrol population are used to establish a reference range. In apreferred embodiment an SLIM-1 concentration is considered as elevatedif the value measured is above the 90%-percentile of the referencerange. In further preferred embodiments an SLIM-1 concentration isconsidered as elevated if the value measured is above the95%-percentile, the 96%-percentile, the 97%-percentile or the97.5%-percentile of the reference range.

In one embodiment the control sample will be an internal control sample.In this embodiment serial samples are obtained from the individual underinvestigation and the marker levels are compared. This may for examplebe useful in assessing the efficacy of therapy.

The method according to the present invention is based on a liquidsample which is obtained from an individual and on the measurement ofSLIM-1 in such sample. An “individual” as used herein refers to a singlehuman or non-human organism. Thus, the methods and compositionsdescribed herein are applicable to both human and veterinary disease.Preferably the individual is a human being.

The SLIM Proteins, Especially SLIM-1

The protein sequences of SLIM-1, SLIM-2 and SLIM-3 each contain 4complete LIM domains and the second half of a fifth LIM domain (FHL,four and a half LIM protein). SLIM-1 has a molecular weight of 36 kD andconsists of 280 amino acids (cf.: SEQ ID NO: 1).

Originally, the LIM protein family was named for the initials of thethree identified transcription factors in which the LIM sequence wasfirst seen: lin-11 (Freyd, G., et al., Nature 344 (1990) 876-879), isle(Karisson, O., et al., Nature 344 (1990) 879-882), and mec-3 (Way, J. C.and Chalfie, M., Cell 54 (1988) 5-16). LIM proteins are involved in awide range of cellular functions like transcription, oncogenictransformation, signal-transduction and in cell adhesion. This may beachieved through protein-protein interactions as the LIM domain containszinc-finger structures. LIM domains can associate with other LIMdomains, thus forming homo- and heterodimers (Feuerstein, R., et al.,Proc. Natl. Acad. Sci. USA 91 (1994) 10655-10659).

SLIM-1/FHL-1 was reported to localize to focal adhesions in skeletalmyoblasts, where it promotes integrin-dependent cell spreading andmigration (Robinson et al., Am. J. Physiol. 284 (2003) C681). Recentlyyeast two-hybrid screening of a human skeletal muscle library identifiedmyosin-binding protein C (MyBP-C) as an FHL-1 binding partner andhypothesized a role of FHL-1 as a regulator of MyBP-C activity andsarcomere assembly (McGrath, M. J., et al., J. Biol. Chem. 281 (2006)7666-7683). The pattern of expression of FHL-1 suggests an importantrole for FHL-1 in the heart during embryonic development (Chu, P. H., etal., Mech. Dev. 95 (200) 259-265) as well as during periods of postnatalskeletal muscle growth.

FHL-1 mRNA levels are found to increase during skeletal musclehypertrophy, induced by stretch (Morgan, M. J., et al., Biochem.Biophys. Res. Commun. 212 (1995) 840-846). FHL-1 mRNA levels declineduring denervation-induced atrophy (Loughna, P. T., et al., Mol. CellBiol. Res. Commun. 3 (2000) 136-140).

Discordant studies report either upregulation or downregulation ofSLIM-1 mRNA in human failing hearts, respectively. FHL-1 m-RNAexpression is reported to be increased in hypertrophied human hearts(Hwang, D. M., et al., Circulation 96 (1997) 4146-4203; Hwang, D. M., etal., Genomics 66 (2000) 1-14; Lim, D.-S., et al., J. Am. Coll. Cardiol.38 (2001) 1175-1180). In contrast, Loughna, P. T., et al., Mol. Cell.Biol. Res. Commun. 3 (2000) 136-140, and Zimmermann, R., et al.,Circulation 100, Suppl. 1 (1999) 565 report a decrease in FHL-1expression. Yang, J., et al., (Circulation 102 (2000) 3046-3052) reportthat in tissue derived from human failing heart both the levels ofSLIM-1 m-RNA and of SLIM-1 protein are reduced.

US 2006/0094038 describes the differential gene expression of numerousgenes, comprising FHL-1 (upregulated) and FHL-2 (downregulated) in thecontext of the diagnosis of an individual's susceptibility to heartfailure.

Furthermore, differential FHL-1 m-RNA expression has been described inmicroarray studies on skin, neural, hematopoietic, and embryonic stemcell populations, suggesting a more widespread role of FHL-1 in diversestem and progenitor cell populations (Ramalho-Santos M., et al., Science298 (2002) 597-600; Tumbar, T., et al., Science 303 (2004) 359-363).

In addition several patent applications deal with tumor diagnosis byanalyzing the differential expression of FHL-1. US 2005/0037389discloses that numerous genes, one of which is FHL-1, may be used forthe diagnosis of uterine serous papillary carcinomas and ovarian serouspapillary tumors. US 2005/0048535 deals with a candidate gene listincluding FHL-1 in association with primary ovarian serous papillarytumors. US2004/0029151 describes genetic profiling of prostate cancerand amongst many other differentially expressed genes mentions SLIM-1.WO 2006/112867 relates to the diagnosis of the aggressiveness ofpapillary renal cell carcinoma genetic by genetic profiling and amongstmany other differentially expressed genes mentions SLIM-1.

WO 2004/092410 describes the differential expression of SLIM-1 in thecontext of rheumatoid arthritis or osteoarthritis, respectively.

Multiple sclerosis is yet a further disease for which an association(upregulation) with the expression of the FHL-1 gene is reported (US2004/0018522 and US 2004/0156826).

It thus appears that in the art the gene expression for SLIM-1 hasextensively been studied by analyzing the corresponding m-RNA levels.These studies have not lead to a clear picture since contradictory datahave been reported. It also appears that no data on the level of theSLIM-1 protein in connection with heart failure in the circulation havebeen shown so far.

Preferably the marker SLIM-1 is specifically measured from a liquidsample by use of a specific binding agent.

A specific binding agent is, e.g., a receptor for SLIM-1, a lectinbinding to SLIM-1 or an antibody to SLIM-1. A specific binding agent hasat least an affinity of 10⁷ l/mol for its corresponding target molecule.The specific binding agent preferably has an affinity of 10⁸ l/mol oreven more preferred of 10⁹ l/mol for its target molecule. As the skilledartisan will appreciate the term specific is used to indicate that otherbiomolecules present in the sample do not significantly bind to thebinding agent specific for SLIM-1. Preferably, the level of binding to abiomolecule other than the target molecule results in a binding affinitywhich is only 10% or less, more preferably only 5% or less of theaffinity to the target molecule, respectively. A preferred specificbinding agent will fulfill both the above minimum criteria for affinityas well as for specificity.

A specific binding agent preferably is an antibody reactive with SLIM-1.The term antibody refers to a polyclonal antibody, a monoclonalantibody, antigen binding fragments of such antibodies, single chainantibodies as well as to genetic constructs comprising the bindingdomain of an antibody.

Any antibody fragment retaining the above criteria of a specific bindingagent can be used. Antibodies are generated by state of the artprocedures, e.g., as described in Tijssen (Tijssen, P., Practice andtheory of enzyme immunoassays, Elsevier Science Publishers B.V.,Amsterdam (1990), the whole book, especially pages 43-78). In addition,the skilled artisan is well aware of methods based on immunosorbentsthat can be used for the specific isolation of antibodies. By thesemeans the quality of polyclonal antibodies and hence their performancein immunoassays can be enhanced (Tijssen, P., supra, pages 108-115).

For the achievements as disclosed in the present invention polyclonalantibodies raised in rabbits may be used. However, clearly alsopolyclonal antibodies from different species, e.g., rats, goats orguinea pigs, as well as monoclonal antibodies can be used. Sincemonoclonal antibodies can be produced in any amount required withconstant properties, they represent ideal tools in development of anassay for clinical routine. The generation and the use of monoclonalantibodies to SLIM-1 in a method according to the present invention,respectively, represent yet other preferred embodiments.

As the skilled artisan will appreciate now, that SLIM-1 has beenidentified as a marker which is useful in the assessment of HF;alternative ways may be used to reach a result comparable to theachievements of the present invention. For example, alternativestrategies to generate antibodies may be used. Such strategies compriseamongst others the use of synthetic or recombinant peptides,representing a clinically relevant epitope of SLIM-1 for immunization.Alternatively, DNA immunization also known as DNA vaccination may beused.

For measurement the liquid sample obtained from an individual isincubated with the specific binding agent for SLIM-1 under conditionsappropriate for formation of a binding agent SLIM-1-complex. Suchconditions need not be specified, since the skilled artisan without anyinventive effort can easily identify such appropriate incubationconditions. The amount of binding agent SLIM-1-complex is measured andused in the assessment of HF. As the skilled artisan will appreciatethere are numerous methods to measure the amount of the specific bindingagent SLIM-1-complex all described in detail in relevant textbooks (cf.,e.g., Tijssen P., supra, or Diamandis, E. P. and Christopoulos, T. K.(eds.), Immunoassay, Academic Press, Boston (1996)).

Preferably SLIM-1 is detected in a sandwich type assay format. In suchassay a first specific binding agent is used to capture SLIM-1 on theone side and a second specific binding agent, which is labeled to bedirectly or indirectly detectable, is used on the other side.Preferably, an antibody to SLIM-1 is used in a qualitative (SLIM-1present or absent) or quantitative (amount of SLIM-1 is determined)immunoassay.

As described in detail in the Examples section, two mouse models havebeen used to identify polypeptides found in heart tissue of experimentalanimals by advanced proteomics methods. However these models did yieldat least partially conflicting data, and, of course tissue data forpolypeptides are not representative to the presence or absence of thesepolypeptides in the circulation. A marker found to be differentiallyexpressed in one model may not be differentially expressed in a secondmodel or even show conflicting data in yet a further model. Even if aprotein may be differentially expressed in tissue this protein in mostcases is not of any diagnostic relevance if measured from a bodilyfluid, because it may not be released to the circulation, may becomefragmented or modified, e.g., upon release from a cell or tissue, maynot be stable in the circulation, may not be measurable in thecirculation, may not be specific for a given disease, etc.

The inventors of the present invention surprisingly are able to detectprotein SLIM-1 in a bodily fluid sample. Even more surprising they areable to demonstrate that the presence of SLIM-1 in such liquid sampleobtained from an individual can be correlated to HF. No tissue and nobiopsy sample is required to make use of the marker SLIM-1 in theassessment of HF. Measuring the level of protein SLIM-1 is consideredvery advantageous in the field of HF.

In a preferred embodiment the method according to the present inventionis practiced with serum as liquid sample material. In a furtherpreferred embodiment the method according to the present invention ispracticed with plasma as liquid sample material. In a further preferredembodiment the method according to the present invention is practicedwith whole blood as liquid sample material.

In a further preferred embodiment, the present invention relates to useof protein SLIM-1 as a marker molecule in the assessment of heartfailure from a liquid sample obtained from an individual.

The ideal scenario for diagnosis would be a situation wherein a singleevent or process would cause the respective disease as, e.g., ininfectious diseases. In all other cases correct diagnosis can be verydifficult, especially when the etiology of the disease is not fullyunderstood as is the case of HF. As the skilled artisan will appreciate,no biochemical marker in the field of HF is diagnostic with 100%specificity and at the same time 100% sensitivity for a certaindiagnostic question. Rather, biochemical markers are used to assess witha certain likelihood or predictive value an underlying diagnosticquestion. The skilled artisan is fully familiar with themathematical/statistical methods that routinely are used to calculate arelative risk or likelihood for the diagnostic question to be assessed.In routine clinical practice various clinical symptoms and biologicalmarkers are generally considered together by a physician in thediagnosis, treatment, and management of the underlying disease.

Preferably in a further preferred embodiment of the present inventionthe method for assessment of HF is performed by measuring theconcentration of SLIM-1 and of one or more other marker and by using theconcentration of SLIM-1 and of the one or more other marker in theassessment of HF.

In the assessment of HF the marker SLIM-1 will aid the physician in oneor more of the following aspects: to assess an individual's risk forheart failure or to assess a patient having heart failure, e.g., withthe intention to identify the stage of heart failure, to differentiatebetween acute and chronic heart failure, to judge the risk of diseaseprogression, to provide guidance in selecting an appropriate therapy, tomonitor a patient's response to therapy, and to monitor the course ofdisease, i.e., in the follow-up of HF patients.

Screening (Assessment Whether Individuals are at Risk for DevelopingHeart Failure)

In a preferred embodiment the present invention relates to an in vitromethod for assessing whether an individual is at risk for developingheart failure comprising the steps of measuring in a sample theconcentration of the marker SLIM-1, of optionally measuring in thesample the concentration of one or more other marker(s) of heartfailure, and of assessing said individual's risk for developing heartfailure by comparing the concentration for SLIM-1 and optionally theconcentration(s) determined for the optionally one or more othermarker(s) to the concentration of this marker or these markers to its ortheir reference value(s).

Screening in the sense of the present invention relates to the unbiasedassessment of individuals regarding their risk for developing heartfailure. Whereas such screening may in theory be performed on anysample, in clinical practice such screening option will usually be givento individuals somehow at risk for development of heart failure. Asdiscussed above, such individuals may clinically be asymptomatic, i.e.,they have no signs or symptoms of HF. In one preferred embodiment,screening for HF will be given to individuals at risk of developingheart failure, e.g. falling into the stages A or B as defined by theACC/AHA practice guidelines.

As mentioned above, heart failure is one of the most prevalent, costlyand life-threatening diseases in developed countries. Because of itshigh prevalence and its long asymptomatic phase identification ofindividuals at risk for developing HF would be of utmost importance tointervene in and if possible to interrupt the course of disease. Withouta very early risk assessment, prevention of disease progression from theasymptomatic state into a symptomatic phase of HF appears impossible.

The risk for heart failure is assessed by mathematical/statisticalmethods fully known and understood by the skilled artisan. Preferably anindividual's risk for heart failure is expressed in relative terms andgiven as the so-called relative risk (=RR). In order to calculate suchRR for heart failure an individual's value for SLIM-1 is compared to thevalues established for SLIM-1 in a reference population, preferablyhealthy individuals not developing heart failure. Also preferred theassessment of such RR for heart failure is based on a group ofindividuals that have developed heart failure within the study period,preferably within one or also preferred within two years, and a group ofindividuals that did not develop heart failure in the same study period.

In another preferred embodiment the present invention relates to the useof the marker SLIM-1 in the screening for heart failure. As the skilledartisan knows the term “use as a marker” implies that the concentrationof a marker molecule is quantified by appropriate means and that valuemeasured for such marker is then used to indicate, i.e. to mark, thepresence or absence of a disease or clinical condition. Appropriatemeans for quantitation for example are specific binding agents, likeantibodies.

Preferably the screening for HF will be performed in individualssuspected to be at risk of future heart failure. Patients at risk offuture heart failure in this sense are patients diagnosed withhypertension, atherosclerotic disease, diabetes, obesity and metabolicsyndrome. Preferably the risk for future heart failure is assessed withindividuals suffering from hypertension, atherosclerotic disease,diabetes, and/or metabolic syndrome.

Also preferred is the use of the marker SLIM-1 in assessing the risk forfuture heart failure for an individual in stage B according to theACC/AHA practice guidelines, i.e., an individual exhibiting a structuralchange at the heart but not showing symptoms of heart failure.

In a further preferred embodiment the present invention relates to theuse of SLIM-1 as one marker of a HF marker combination for HF screeningpurposes.

In the screening setting an elevated level of SLIM-1 is a positiveindicator for an individual's increased risk to develop heart failure.

Staging of Patients

In a preferred embodiment the present invention relates to an in vitromethod aiding in the staging of heart failure patients, comprising thesteps of a) measuring in a sample the concentration of the markerSLIM-1, of b) optionally measuring in the sample the concentration ofone or more other marker(s) of heart failure, and staging heart failureby comparing the concentration determined in step (a) and optionally theconcentration(s) determined in step (b) to the concentration of thismarker or these markers to its or their reference value(s). Preferablythe level of marker SLIM-1 is used as an aid in classifying theindividuals investigated into the groups of individuals that areclinically “normals” (i.e., individuals in stage A according to theACA/ACC classification), asymptomatic patients having structural heartdisease (stage B according to the ACA/ACC classification) and the groupof patients having heart failure (i.e., patients in stage C or stage Daccording to the ACA/ACC classification).

Differentiation Between an Acute Cardiac Event and Chronic CardiacDisease

In a preferred embodiment the present invention relates to an in vitromethod aiding in the differential diagnosis between an acute cardiacevent and chronic cardiac disease, comprising the steps of measuring ina sample the concentration of the marker SLIM-1, of optionally measuringin the sample the concentration of one or more other marker(s) of heartfailure, and establishing a differential diagnosis between an acutecardiac event and chronic cardiac disease by comparing the concentrationdetermined in step (a) and optionally the concentration(s) determined instep (b) to the concentration of this marker or these markers to its ortheir reference value(s).

The person skilled in the art is familiar with the meanings of “acutecardiac event” and of “chronic cardiac disease”.

Preferably, an “acute cardiac event” relates to an acute condition,disease or malfunction of the heart, particularly to acute heartfailure, e.g., myocardial infarction (MI) or arrhythmia. Depending onthe extent of an MI, it may be followed by LVD and CHF.

Preferably, a “chronic cardiac disease” is a weakening of heartfunction, e.g., due to ischemia of the heart, coronary artery disease,or previous, particularly small, myocardial infarction(s) (possiblyfollowed by progressing LVD). It may also be a weakening due toinflammatory diseases, heart valve defects (e.g., mitral valve defects),dilatative cardiomyopathy, hypertrophic cardiomyopathy, heart rhythmdefects (arrhythmias), and chronic obstructive pulmonary disease. Thus,it is clear that a chronic cardiac disease may also include patients whohad suffered from an acute coronary syndrome, e.g., MI, but who arepresently not suffering from an acute cardiac event.

It is important to differentiate between an acute cardiac event andchronic cardiac disease, because an acute cardiac event and chroniccardiac disease may require quite different treatment regimens. Forexample, for a patient presenting with acute myocardial infarction earlytreatment for reperfusion may be of utmost importance. Whereas atreatment for reperfusion performed on a patient with chronic heartfailure at best is of no or only little harm to this patient.

In a further preferred embodiment according to the present invention themarker SLIM-1 is used in the differential diagnosis of acute and chronicheart failure.

Assessing the Risk of Disease Progression

In a preferred embodiment the present invention relates to an in vitromethod for assessing an HF-patient's risk for disease progression,comprising the steps of measuring in a sample the concentration of themarker SLIM-1, of optionally measuring in the sample the concentrationof one or more other marker(s) of heart failure, and of establishingsaid individual's risk for disease progression by comparing theconcentration for SLIM-1 and optionally the concentration(s) determinedfor the optionally one or more other marker(s) to the concentration ofthis marker or these markers to its or their reference value(s).

At present it is very difficult to assess or to even predict with areasonable likelihood whether a patient diagnosed with HF has a more orless stable status or whether the disease will progress and thepatient's health status as result is likely to worsen. Severity andprogression of heart failure is clinically usually established byassessing the clinical symptoms or by identification of adverse changesby using imaging technologies such as echocardiography. In oneembodiment the worsening of heart failure is established by monitoringthe left ventricular ejection fraction (LVEF). A deterioration in LVEFby 5% or more is considered as disease progression.

In a further preferred embodiment the present invention thereforerelates to the use of the marker SLIM-1 in assessing the risk of diseaseprogression for a patient suffering from HF. In the assessment ofdisease progression for patients suffering from HF an elevated level ofSLIM-1 is an indicator for an increased risk of disease progression inthe early stages of HF, whereas a decreased level of SLIM-1 isindicative for end-stage heart failure.

Guidance in Selecting an Appropriate HF Therapy

In a preferred embodiment the present invention relates to an in vitromethod, aiding in the selection of an appropriate HF-therapy, comprisingthe steps of measuring in a sample the concentration of the markerSLIM-1, of optionally measuring in the sample the concentration of oneor more other marker(s) of heart failure, and of selecting anappropriate therapy by comparing the concentration for SLIM-1 andoptionally the concentration(s) determined for the optionally one ormore other marker(s) to the concentration of this marker or thesemarkers to its or their reference value(s).

It is expected that the marker SLIM-1 will be of help in aiding thephysician to select the most appropriate treatment regimen from thevarious treatment regimens at hand in the area of heart failure.

In a further preferred embodiment therefore relates to the use of themarker SLIM-1 in selecting a treatment regimen for a patient sufferingfrom HF.

Monitor a Patient's Response to Therapy

In a preferred embodiment the present invention relates to an in vitromethod for monitoring a patient's response to HF-therapy, comprising thesteps of a) measuring in a sample the concentration of the markerSLIM-1, of b) optionally measuring in the sample the concentration ofone or more other marker(s) of heart failure, and of monitoring apatient's response to HF-therapy by comparing the concentrationdetermined in step (a) and optionally the concentration(s) determined instep (b) to the concentration of this marker or these markers to its ortheir reference value(s).

Alternatively the above method for motoring a patient's response totherapy can be practiced by establishing the pre- and post-therapeuticmarker level for SLIM-1 and for the optionally one or more other markerand by comparing the pre- and the post-therapeutic marker level(s).

The diagnosis of heart failure is clinically established. According tothe present invention HF is considered clinically established if apatient meets the criteria of stages C or D as defined by the ACC/AHApractice guidelines. According to these guidelines stage C refers topatients with structural heart disease and with prior or currentsymptoms of heart failure. Patients in stage D are those patients withrefractory heart failure that require specialized interventions.

As indicated further above the values measured for NT-proBNP are highlycorrelated to the severity of heart failure. However, both BNP andNT-proBNP appear to be not ideal in monitoring a patient's response totherapy, cf. e.g., Beck-da-Silva, L., et al., Congest. Heart. Fail. 11(2005) 248-253, quiz 254-255.

The marker SLIM-1 appears to be appropriate to monitor a patient'sresponse to therapy. The present invention thus also relates to the useof SLIM-1 in monitoring a patient's response to therapy. In thatdiagnostic area the marker SLIM-1 can also be used for establishing abaseline value before therapy and to measure SLIM-1 at one time-point orseveral time-points after therapy. In the follow-up of HF patients anelevated level of SLIM-1 is a positive indicator for an effectivetreatment of HF.

Marker Combination

Biochemical markers can either be determined individually or, in apreferred embodiment of the invention, they can be measuredsimultaneously using a chip- or a bead-based array technology. Theconcentrations of the biomarkers are then interpreted independentlyusing an individual cut-off for each marker or they are combined forinterpretation, i.e., they form a marker combination.

As the skilled artisan will appreciate the step of correlating a markerlevel to a certain likelihood or risk can be performed and achieved indifferent ways. Preferably the values measured for the marker SLIM-1 andthe one or more other marker(s), are mathematically combined and thecombined value is correlated to the underlying diagnostic question.Marker values may be combined with the measurement of SLIM-1 by anyappropriate state of the art mathematical method.

Preferably the mathematical algorithm applied in the combination ofmarkers is a logistic function. The result of applying such mathematicalalgorithm or such logistical function preferably is a single value.Dependent on the underlying diagnostic question such value can easily becorrelated to e.g., the risk of an individual for heart failure or toother intended diagnostic uses helpful in the assessment of patientswith HF. In a preferred way such logistic function is obtained by a)classification of individuals into the groups, e.g., into normals,individuals at risk for heart failure, patients with acute or chronicheart failure and so on, b) identification of markers which differsignificantly between these groups by univariate analysis, c) logisticregression analysis to assess the independent discriminative values ofmarkers useful in assessing these different groups and d) constructionof the logistic function to combine the independent discriminativevalues. In this type of analysis the markers are no longer independentbut represent a marker combination.

In a preferred embodiment the logistic function used for combining thevalues for SLIM-1 and the value of at least one further marker isobtained by a) classification of individuals into the groups of normalsand individuals at risk of heart failure, respectively, b) establishingthe values for SLIM-1 and the value of the at least one further markerc) performing logistic regression analysis and d) construction of thelogistic function to combine the marker values for SLIM-1 and the valueof the at least one further marker.

A logistic function for correlating a marker combination to a diseasepreferably employs an algorithm developed and obtained by applyingstatistical methods. Appropriate statistical methods are, e.g.,Discriminant analysis (DA) (i.e., linear-, quadratic-, regularized-DA),Kernel Methods (i.e., SVM), Nonparametric Methods (i.e.,k-Nearest-Neighbor Classifiers), PLS (Partial Least Squares), Tree-BasedMethods (i.e., Logic Regression, CART, Random Forest Methods,Boosting/Bagging Methods), Generalized Linear Models (i.e., LogisticRegression), Principal Components based Methods (i.e., SIMCA),Generalized Additive Models, Fuzzy Logic based Methods, Neural Networksand Genetic Algorithms based Methods. The skilled artisan will have noproblem in selecting an appropriate statistical method to evaluate amarker combination of the present invention and thereby to obtain anappropriate mathematical algorithm. Preferably the statistical methodemployed to obtain the mathematical algorithm used in the assessment ofheart failure is selected from DA (i.e., Linear-, Quadratic-,Regularized Discriminant Analysis), Kernel Methods (i.e., SVM),Nonparametric Methods (i.e., k-Nearest-Neighbor Classifiers), PLS(Partial Least Squares), Tree-Based Methods (i.e., Logic Regression,CART, Random Forest Methods, Boosting Methods), or Generalized LinearModels (i.e., Logistic Regression). Details relating to thesestatistical methods are found in the following references: Ruczinski,I., et al., J. of Computational and Graphical Statistics 12 (2003)475-511; Friedman, J. H., J. of the American Statistical Association 84(1989) 165-175; Hastier T., et al., The Elements of StatisticalLearning, Springer Verlag (2001); Breiman, L., et al., Classificationand regression trees, Wadsworth International Group, California (1984);Breiman, L., Machine Learning 45 (2001) 5-32; Pepe, M. S., TheStatistical Evaluation of Medical Tests for Classification andPrediction, Oxford Statistical Science Series, 28, Oxford UniversityPress (2003); and Duda, R. O., et al., Pattern Classification, JohnWiley & Sons, Inc., 2nd ed. (2001).

It is a preferred embodiment of the invention to use an optimizedmultivariate cut-off for the underlying combination of biologicalmarkers and to discriminate state A from state B, e.g., normals andindividuals at risk for heart failure, HF patients responsive to therapyand therapy failures, patients having an acute heart failure and HFpatients having chronic heart failure, HF patients showing diseaseprogression and HF patients not showing disease progression,respectively.

The area under the receiver operator curve (=AUC) is an indicator of theperformance or accuracy of a diagnostic procedure. Accuracy of adiagnostic method is best described by its receiver-operatingcharacteristics (ROC) (see especially Zweig, M. H., and Campbell, G.,Clin. Chem. 39 (1993) 561-577). The ROC graph is a plot of all of thesensitivity/specificity pairs resulting from continuously varying thedecision thresh-hold over the entire range of data observed.

The clinical performance of a laboratory test depends on its diagnosticaccuracy, or the ability to correctly classify subjects into clinicallyrelevant subgroups. Diagnostic accuracy measures the test's ability tocorrectly distinguish two different conditions of the subjectsinvestigated. Such conditions are for example, health and disease ordisease progression versus no disease progression.

In each case, the ROC plot depicts the overlap between the twodistributions by plotting the sensitivity versus 1—specificity for thecomplete range of decision thresholds. On the y-axis is sensitivity, orthe true-positive fraction [defined as (number of true-positive testresults)/(number of true-positive+number of false-negative testresults)]. This has also been referred to as positivity in the presenceof a disease or condition. It is calculated solely from the affectedsubgroup. On the x-axis is the false-positive fraction, or 1—specificity[defined as (number of false-positive results)/(number oftrue-negative+number of false-positive results)]. It is an index ofspecificity and is calculated entirely from the unaffected subgroup.Because the true- and false-positive fractions are calculated entirelyseparately, by using the test results from two different subgroups, theROC plot is independent of the prevalence of disease in the sample. Eachpoint on the ROC plot represents a sensitivity/1—specificity paircorresponding to a particular decision threshold. A test with perfectdiscrimination (no overlap in the two distributions of results) has anROC plot that passes through the upper left corner, where thetrue-positive fraction is 1.0, or 100% (perfect sensitivity), and thefalse-positive fraction is 0 (perfect specificity). The theoretical plotfor a test with no discrimination (identical distributions of resultsfor the two groups) is a 45° diagonal line from the lower left corner tothe upper right corner. Most plots fall in between these two extremes.(If the ROC plot falls completely below the 45° diagonal, this is easilyremedied by reversing the criterion for “positivity” from “greater than”to “less than” or vice versa.) Qualitatively, the closer the plot is tothe upper left corner, the higher the overall accuracy of the test.

One convenient goal to quantify the diagnostic accuracy of a laboratorytest is to express its performance by a single number. The most commonglobal measure is the area under the ROC plot (AUC). By convention, thisarea is always ≧0.5 (if it is not, one can reverse the decision rule tomake it so). Values range between 1.0 (perfect separation of the testvalues of the two groups) and 0.5 (no apparent distributional differencebetween the two groups of test values). The area does not depend only ona particular portion of the plot such as the point closest to thediagonal or the sensitivity at 90% specificity, but on the entire plot.This is a quantitative, descriptive expression of how close the ROC plotis to the perfect one (area=1.0).

The overall assay sensitivity will depend on the specificity requiredfor practicing the method disclosed here. In certain preferred settingsa specificity of 75% may be sufficient and statistical methods andresulting algorithms can be based on this specificity requirement. Inone preferred embodiment the method used to assess individuals at riskfor heart failure is based on a specificity of 80%, of 85%, or alsopreferred of 90% or of 95%.

As discussed above, the marker SLIM-1 aids in assessing an individualsrisk of developing heart failure as well as in the further in vitrodiagnostic assessment of a patient having heart failure. A preferredembodiment accordingly is the use of SLIM-1 as a marker molecule in theassessment of heart failure.

The use of a marker combination comprising SLIM-1 and one or more othermarker(s) of HF in the assessment of HF patients or in the assessment ofindividuals at risk for HF represents a further preferred embodiment ofthe present invention. In such marker combination the one or more othermarker(s) preferably is selected from the group consisting of anatriuretic peptide marker, a cardiac troponin marker, and a marker ofinflammation.

The one or more preferred selected other HF marker(s) with which themeasurement of SLIM-1 may be combined preferably is or are selected fromthe group consisting of a natriuretic peptide marker, a cardiac troponinmarker, and a marker of inflammation. These preferred other markerswhose measurement(s) preferably are combined with the measurement ofSLIM-1 or which form part of the HF marker combination comprisingSLIM-1, respectively, are discussed in more detail below.

Natriuretic Peptide Marker

A natriuretic peptide marker in the sense of the present invention iseither a marker selected from the atrial natriuretic peptide (ANP)family or a marker selected from the brain natriuretic peptide (BNP)family.

The polypeptide markers in either the atrial natriuretic peptide familyor in the brain natriuretic peptide family are derived from thepreproforms of the corresponding active hormones.

Preferred natriuretic peptide markers according to the present inventionare NT-proANP, ANP, NT-proBNP, BNP, and immunologically detectablephysiological fragments thereof. As the skilled artisan readilyappreciates, the immunologically detectable fragment has to comprise atleast one epitope allowing for the specific detection of suchphysiological fragment. A physiological fragment is a fragment asnaturally present in an individual's circulation.

The markers in both the natriuretic peptide families represent fragmentsof the corresponding pro-hormones, i.e., proANP and proBNP,respectively. Since similar considerations apply for both families, onlythe BNP marker family shall be described in some detail. The pro-hormoneof the BNP family, i.e., proBNP consists of 108 amino acids. proBNP iscleaved into the 32 C-terminal amino acids (77-108) representing thebiologically active hormone BNP and the N-terminal amino acids 1-76called N-terminal proBNP (or NT-proBNP). BNP, N-terminal proBNP (1-76)as well as further breakdown products (Hunt, P. J., et al., Biochem.Biophys. Res. Com. 214 (1995) 1175-1183) circulate in blood. Whether thecomplete precursor molecule (proBNP 1-108) also occurs in the plasma isnot completely resolved. It is however described (Hunt, P. J., et al.,Peptides 18 (1997) 1475-1481) that a low release of proBNP (1-108) inplasma is detectable but that due to the very quick partial breakdown atthe N-terminal end some amino acids are absent. Today it is generallyaccepted that e.g., for NT-proBNP the central portion of the molecule,residing in between the amino acids 10 to 50 represents aphysiologically rather stable part. NT-proBNP molecules comprising thiscentral part of NT-proBNP can be reliably measured from bodily fluids.Detailed disclosure relating to methods based on the immunologicaldetection of this central part of the NT-proBNP molecule is given in WO00/45176 and the reader is referred thereto for details. It may be offurther advantage to measure only a certain subfraction of NT-proBNP forwhich the term native NT-proBNP has been proposed. Detailed disclosurerelating to this subfraction of NT-proBNP is found in WO 2004/099253.The artisan will find all necessary instructions there. Preferably theNT-proBNP measured is or corresponds to the NT-proBNP as measured withthe ELECSYS NT-proBNP assay from Roche Diagnostics, Germany.

Preanalytics are robust with NT-proBNP, which allows easy transportationof the sample to a central laboratory (Mueller, T., et al. Clin. Chem.Lab. Med. 42 (2004) 942-944). Blood samples can be stored at roomtemperature for several days or may be mailed or shipped withoutrecovery loss. In contrast, storage of BNP for 48 hours at roomtemperature or at 4° Celsius leads to a concentration loss of at least20% (Mueller, T., et al., supra; Wu, A. H., et al., Clin. Chem. 50(2004) 867-873).

The brain-derived natriuretic peptide family (especially BNP andNT-proBNP) has been thoroughly investigated in the screening of certainpopulations for HF. The findings with these markers, especially withNT-proBNP are quite encouraging. Elevated values of NT-proBNP even inasymptomatic “patients” are clearly indicative for “heart problems”(Gremmler, B., et al., Exp. Clin. Cardiol. 8 (2003) 91-94). Theseauthors showed that an elevated NT-proBNP indicates the presence of‘cardio-renal distress’ and should prompt referral for furtherinvestigation. In line with several other groups of investigatorsGremmler et al. also find that an abnormal NT-proBNP concentration is anaccurate diagnostic test both for the exclusion of HF in the populationand in ruling out left ventricular dysfunction (=LVD) in breathlesssubjects. The role of negative BNP or NT-proBNP values in ruling out HFor LVD is corroborated by other groups of investigators, cf., e.g.,McDonagh, T. A., et al., Eur. J. Heart Fail. 6 (2004) 269-273 andGustafsson, F., et al., J. Card. Fail. 11, Suppl. 5 (2005) S15-20.

BNP is produced predominantly (albeit not exclusively) in the ventricleand is released upon increase of wall tension. Thus, an increase ofreleased BNP reflects predominantly dysfunctions of the ventricle ordysfunctions which originate in the atria but affect the ventricle,e.g., through impaired inflow or blood volume overload. In contrast toBNP, ANP is produced and released predominantly from the atrium. Thelevel of ANP may therefore predominantly reflect atrial function.

ANP and BNP are the active hormones and have a shorter half-life thantheir respective inactive counterparts, NT-proANP and NT-proBNP. BNP ismetabolised in the blood, whereas NT-proBNP circulates in the blood asan intact molecule and as such is eliminated renally. The in-vivohalf-life of NT-proBNP is 120 min longer than that of BNP, which is 20min (Smith, M. W., et al., J. Endocrinol. 167 (2000) 239-246).

Therefore, depending on the time-course or properties of interest,either measurement of the active or the inactive forms of thenatriuretic peptide can be advantageous.

In the assessment of an individual at risk for heart failure the valuemeasured for SLIM-1 is preferably combined with the value for NT-proANPand/or NT-proBNP. Preferably the value for NT-proBNP is combined withthe value for SLIM-1. Similar considerations apply for selecting anappropriate therapy, judging the risk of disease progression, and tomonitoring the course of disease.

In case SLIM-1 is used in assessing a patient's response to therapy itsmeasurement is preferably combined with the measurement of ANP or BNP.

In case SLIM-1 is used to differentiate between acute and chronic heartfailure the preferred marker combination comprises SLIM-1, ANP or proANPand BNP or proBNP.

Cardiac Troponin Marker

The term cardiac troponin relates to the cardiac isoforms of troponin Iand troponin T. As already indicated above the term marker also relatesto physiological variants of the marker molecule, like physiologicalfragments or complexes. For the cardiac troponin markers theirphysiologically occurring complexes are known to be of diagnosticrelevance and are herewith expressly included.

Troponin T has a molecular weight of about 37.000 Da. The troponin Tisoform that is found in cardiac tissue (cTnT) is sufficiently divergentfrom skeletal muscle TnT to allow for the production of antibodies thatdistinguish both these TnT isoforms. TnT is considered a marker of acutemyocardial damage; cf. Katus, H. A., et al., J. Mol. Cell. Cardiol. 21(1989) 1349-1353; Hamm, C. W., et al., N. Engl. J. Med 327 (1992)146-150; Ohman, E. M., et al., N. Engl. J. Med. 335 (1996) 1333-1341;Christenson, R. H., et al., Clin. Chem. 44 (1998) 494-501; and EP 0 394819.

Troponin I (TnI) is a 25 kDa inhibitory element of the troponin complex,found in muscle tissue. TnI binds to actin in the absence of Ca²⁺,inhibiting the ATPase activity of actomyosin. The TnI isoform that isfound in cardiac tissue (cTnI) is 40% divergent from skeletal muscleTnI, allowing both isoforms to be immunologically distinguished. Thenormal plasma concentration of cTnI is <0.1 ng/ml (4 pM). cTnI isreleased into the bloodstream following cardiac cell death; thus, theplasma cTnI concentration is elevated in patients with acute myocardialinfarction (Benamer, H., et al., Am. J. Cardiol. 82 (1998) 845-850).

The unique cardiac isoforms of troponin I and T allow them to bedistinguished immunologically from the other troponins of skeletalmuscle. Therefore, the release into the blood of troponin I and T fromdamaged heart muscle can be specifically related to damage of cardiactissue. It is nowadays also appreciated by the skilled artisan that thecardiac troponins may be detected from the circulation either in theirfree form or as a part of a complex (cf. e.g., U.S. Pat. No. 6,333,397,U.S. Pat. No. 6,376,206 and U.S. Pat. No. 6,174,686).

In the assessment of an individual at risk for heart failure as well asin the assessment of a patient suffering from heart failure, the valuemeasured for SLIM-1 is preferably combined with the value for cardiacisoform of troponin T and/or troponin I. A preferred cardiac troponinused in combination with the marker SLIM-1 is cardiac troponin T.

Marker of Inflammation

The skilled artisan is familiar with the term marker of inflammation.Preferred markers of inflammation are interleukin-6, C-reactive protein,serum amyloid A and a S100 protein.

Interleukin-6 (IL-6) is a 21 kDa secreted protein that has numerousbiological activities that can be divided into those involved inhematopoiesis and into those involved in the activation of the innateimmune response. IL-6 is an acute-phase reactant and stimulates thesynthesis of a variety of proteins, including adhesion molecules. Itsmajor function is to mediate the acute phase production of hepaticproteins, and its synthesis is induced by the cytokines IL-1 and TNF-α.IL-6 is normally produced by macrophages and T lymphocytes. The normalserum concentration of IL-6 is <5 pg/ml.

C-reactive protein (CRP) is a homopentameric Ca²⁺-binding acute phaseprotein with 21 kDa subunits that is involved in host defense. CRPsynthesis is induced by IL-6, and indirectly by IL-1, since IL-1 cantrigger the synthesis of IL-6 by Kupffer cells in the hepatic sinusoids.The normal plasma concentration of CRP is <3 μg/ml (30 nM) in 90% of thehealthy population, and <10 μg/ml (100 nM) in 99% of healthyindividuals. Plasma CRP concentrations can, e.g., be measured by Serumamyloid A (=SAA) is an acute phase protein of low molecular weight of11.7 kDa. It is predominantly synthesized by the liver in response toIL-1, IL-6 or TNF-α stimulation and is involved in the regulation of theT-cell dependent immune response. Upon acute events the concentration ofSAA increases up to 1000-fold reaching one milligram per milliliter. Itis used to monitor inflammation in diseases as divers as cysticfibrosis, renal graft refection, trauma or infections. In rheumatoidarthritis is has in certain cases been used as a substitute for CRP,but, SAA is not yet as widely accepted.

S100-proteins form a constantly increasing family of Ca²⁺-bindingproteins that today includes more than 20 members. The physiologicallyrelevant structure of S100-proteins is a homodimer but some can alsoform heterodimers with each other, e.g., S100A8 and S100A9. Theintracellular functions range from regulation of proteinphosphorylation, of enzyme activities, or of the dynamics of thecytoskeleton to involvement in cell proliferation and differentiation.As some S100-proteins are also released from cells, extracellularfunctions have been described as well, e.g., neuronal survival,astrocyte proliferation, induction of apoptosis and regulation ofinflammatory processes. S100A8, S100A9, the heterodimer S100A8/A9 andS100A12 have been found in inflammation with S100A8 responding tochronic inflammation, while S100A9, S100A8/A9 and S100A12 are increasedin acute inflammation. S100A8, S100A9, S100A8/A9 and S100A12 have beenlinked to different diseases with inflammatory components including somecancers, renal allograft rejection, colitis and most importantly to RA(Burmeister, G., and Gallacchi, G., Inflammopharmacology 3 (1995)221-230; Foell, D., et al., Rheumatology 42 (2003) 1383-1389). The mostpreferred S100 markers for assessing an individual at risk for HF or apatient having HF e.g., for use in a marker combination according to thepresent invention are S100A8, S100A9, S100A8/A9 heterodimer and S100A12.

sE-selectin (soluble endothelial leukocyte adhesion molecule-1, ELAM-1)is a 115 kDa, type-I transmembrane glycoprotein expressed only onendothelial cells and only after activation by inflammatory cytokines(IL-1β, TNF-α) or endotoxin. Cell-surface E-selectin is a mediator ofthe rolling attachment of leucocytes to the endothelium, an essentialstep in extravasion of leucocytes at the site of inflammation, therebyplaying an important role in localized inflammatory response. SolubleE-selectin is found in the blood of healthy individuals, probablyarising from proteolytic cleavage of the surface-expressed molecule.Elevated levels of sE-selectin in serum have been reported in a varietyof pathological conditions (Gearing, A. J. H. and Hemingway, I., Ann.N.Y. Acad. Sci. 667 (1992) 324-331).

In a preferred embodiment the present invention relates to the use ofSLIM-1 as a marker molecule for HF in combination with one or moremarker molecule(s) for HF in the assessment of HF from a liquid sampleobtained from an individual.

As indicated above, in a preferred method according to the presentinvention the value measured for SLIM-1 is at least combined with thevalue of at least one further marker selected from the group consistingof a natriuretic peptide marker, a cardiac troponin marker, and a markerof inflammation.

In a preferred embodiment the present invention relates to the use ofthe marker combination SLIM-1 and NT-proBNP in the assessment of heartfailure.

In a preferred embodiment the present invention relates to the use ofthe marker combination SLIM-1 and troponin T in the assessment of heartfailure.

In a preferred embodiment the present invention relates to the use ofthe marker combination SLIM-1 and CRP in the assessment of heartfailure.

In a further preferred embodiment the present invention relates to amarker combination comprising the markers SLIM-1, troponin T, NT-proBNPand CRP.

In yet a further preferred embodiment the present invention relates to amarker panel used in a method for assessing HF in vitro by biochemicalmarkers, comprising measuring in a sample the concentration of SLIM-1and of one or more other marker of HF and using the concentrationsdetermined in the assessment of HF.

A marker panel according to the present invention is preferably measuredusing a protein array technique. An array is a collection of addressableindividual markers. Such markers can be spacially addressable, such asarrays contained within microtiter plates or printed on planar surfaceswhere each marker is present at distinct X and Y coordinates.Alternatively, markers can be addressable based on tags, beads,nanoparticles, or physical properties. The microarrays can be preparedaccording to the methods known to the ordinarily skilled artisan (seefor example, U.S. Pat. No. 5,807,522; Robinson, W. H., et al., Nat. Med.8 (2002) 295-301; Robinson, W. H., et al., Arthritis Rheum. 46 (2002)885-893). Array as used herein refers to any immunological assay withmultiple addressable markers. In one embodiment the addressable markersare antigens. In another embodiment the addressable elements areautoantibodies. A microarray is a miniaturized form of an array. Antigenas used herein refers to any molecule that can bind specifically to anantibody. The term autoantibody is well-defined in the art.

In a preferred embodiment the present invention relates to protein arraycomprising the marker SLIM-1 and optionally one or more other marker ofHF.

In a preferred embodiment the present invention relates to a proteinarray comprising the markers SLIM-1 and NT-proBNP.

In a preferred embodiment the present invention relates to a proteinarray comprising the markers SLIM-1 and troponin T.

In a preferred embodiment the present invention relates to a proteinarray comprising the markers SLIM-1 and CRP.

In a further preferred embodiment the present invention relates to aprotein array comprising the markers SLIM-1, troponin T, NT-proBNP andCRP.

In yet a further preferred embodiment the present invention relates to akit comprising the reagents required to specifically measure SLIM-1.Also preferred is a kit comprising the reagents required to specificallymeasure SLIM-1 and the reagents required to measure the one or moreother marker of heart failure that are used together in an HF markercombination.

The following examples, sequence listing and figures are provided to aidthe understanding of the present invention, the true scope of which isset forth in the appended claims. It is understood that modificationscan be made in the procedures set forth without departing from thespirit of the invention.

Example 1 Mouse Models of Heart Failure

1.1: The R9C Mouse Model

It has been reported that an inherited human dilated cardiomyopathyresulted from the conversion of Arg9 to Cys in the human phospholamban(PLN) gene (PLN-R9C) (Schmitt, J. P., et al., Science 299 (2003)1410-1413). The onset of dilated cardiomyopathy in affected patientstypically commenced during adolescence, followed by progressivedeterioration in cardiac function leading to crisis and mortality. Atransgenic mouse model of this mutation showed similar cardiac phenotypeas the affected patients and presented with dilated cardiomyopathy,decreased cardiac contractility, and premature death (Schmitt, 2003,supra).

We established a survival curve for the transgenic mice. The PLN-R9Cmice had a median survival of only ˜20 weeks with fewer than 15%persisting past 24 weeks (FIG. 1 A). The first recorded deaths in thePLN-R9C line are observed at 12 weeks of age, while only one wild-typecontrol mouse died over the 24 week period. Eight weeks is selected asrepresentative time point of ‘early’ stage disease prior to the firstrecorded mortality, while 16 weeks is chosen as it is the midpointbetween 8 and 24 weeks (classic DCM). A detailed analysis of thepathology of isolated hearts shows evidence of ventricle and atriaenlargement even at 8 weeks of age in the PLN-R9C mice. Cross-sectionsof isolated cardiac muscle (obtained from wild-type and PLN-R9C micefollowed by hematoxylin and eosin staining also shows evidence of leftventricular dilatation, or thinning of the ventricular wall, in thetransgenic animals beginning at 8 weeks, with continued progression ofdilatation with age.

Functional cardiac measurements are performed by echocardiography on the8, 16 and 24 week old male mice (summarized in Table 1).Echocardiography measurements of the thickness of the anterior andposterior wall show that the R9C mice have significant dilatation at 8weeks, which continues to deteriorate throughout the lifespan of themice. Contractility, as assessed by cardiac shortening (FIG. 1 B), isalso slightly, but significantly, reduced at 8 weeks, while a morepronounced decrease is clearly evident by 16 weeks. Female mice analyzedshow identical findings as the males (data not shown).

TABLE 1 Echocardiographic and hemodynamic parameters in wildtype and R9Cmice at 8, 16, and 24 weeks in male mice. WT R9C WT R9C WT R9C Age 8 wks8 wks 16 wks 16 wks 24 wks 24 wks Gender M M M M M M HR (bpm) 560 ± 6 567 ± 5   569 ± 5  552 ± 15  565 ± 9  502 ± 15*  AW (mm) 0.66 ± 0.010.60 ± 0.01* 0.70 ± 0.01 0.58 ± 0.01* 0.71 ± 0.01 0.57 ± 0.01* PW (mm)0.66 ± 0.01 0.61 ± 0.01* 0.70 ± 0.01 0.59 ± 0.01* 0.71 ± 0.01 0.57 ±0.01* LVEDD (mm) 3.82 ± 0.05 4.01 ± 0.03* 3.92 ± 0.07 5.01 ± 0.06* 3.99± 0.05 5.48 ± 0.08* LVESD (mm) 1.82 ± 0.05 2.13 ± 0.04* 1.84 ± 0.06 3.36± 0.09* 1.89 ± 0.03 4.23 ± 0.09* FS (%) 52.7 ± 0.9  47.6 ± 1.2*  53.1 ±0.7  32.9 ± 1.9*  52.9 ± 1.5  22.6 ± 2.1*  VCFc (circ/s) 10.5 ± 0.2  9.1± 0.2* 10.5 ± 0.1  7.0 ± 0.5* 10.9 ± 0.3  5.1 ± 0.5* PAVc (cm/s) 102.4 ±2.4  97.8 ± 2.6  110.1 ± 3.7  85.3 ± 3.2*  111.3 ± 2.9  73.6 ± 3.1*  AVA(m/s2) 65.7 ± 1.3  60.6 ± 1.6   66 ± 3.2 47.9 ± 2.5*  67.1 ± 3.1   40 ±2.2* Samples (n) 6 9 6 9 5 5

Values in Table 1 are mean±SEM. Symbols used in Table 1: HR=Heart Rate;AW, PW=Anterior and Posterior Wall Thickness (Left Ventricle); LVEDD,LVESD=Left Ventricular End Diastolic and Systolic Dimension,respectively; FS=Fractional Shortening=(LVEDD−LVESD)/LVEDD×100%;ETC=Ejection Time corrected for HR; VCFC=Velocity of CircumferentialShortening corrected for HR=FS/ETC; PAVC=Peak Aortic Velocity correctedfor HR; E-wave=Early-filling transmitral diastolic wave; LVESP,LVEDP=Left Ventricular End Systolic and Diastolic Pressure;+dP/dtmax=Maximum positive 1st derivative of the left ventricularpressure; −P/dtmax=Maximum negative 1st derivative of the leftventricular pressure; AVA=aortic velocity acceleration(PAVc/Acceleration Time); *P<0.05 compared with WT.

1.2: The Aortic Banding (AB) Mouse Model

In this mouse model pressure-overload caused by aortic banding (AB)induces cardiac-hypertrophy.

By surgical intervention pressure-overload is performed in C57BL mice.The coarction of the ascending aorta (known as aortic banding) inducescardiac hypertrophy and growth of the myocardial muscle, especially inthe left ventricle as a primary response to coarction of the aorta. Inthe later stages of this mouse model the heart becomes hypertrophic andfinally dilated. This model is well characterized and has proven to behighly reproducible with a low mortality rate of 10-15% or less based onexperience. After coarction this animal model allows for evaluating theprogress of development of left ventricular hypertrophy and heartfailure in response to hemodynamic stress.

Briefly C57BL mice are anesthetized with mixed Ketamine (90 mg/kg) andRompun (10 mg/Kg) and the aorta is ligated using 25-gauge needle. Shamoperated mice undergo the same surgical procedure, except that theligation is not tightened against the needle.

Experimental Time Points

To examine the primary hypertrophic response as well as the dilatedresponse at a later stage the, banded animals and sham-operated controlsare sacrificed at one, two, four, and eight weeks post intervention.Cardiac function and the development of hypertrophy are assessed byechocardiographic analysis and confirmed post mortem by examining thehistology. Table 2 shows an overview over the cardiac function evaluatedat the various time points by echocardiography. Details on theechocardiographic parameters given in Table 2 are known to the artisanand can e.g., be found in Asahi, M., et al., Proc. Natl. Acad. Sci. USA101 (2004) 9199-9204, and Oudit, G. Y., et al., Nat. Med. 9 (2003)1187-1194.

TABLE 2 Parameter 2 wk sham 2 wk AB 4 wk sham 4 wk AB 8 wk sham 8 wk ABHeart rate (bpm) 271.6 ± 31.2 286.3 ± 39.1 275.3 ± 25.8 276.5 ± 28.1255.5 ± 23.9 310.8 ± 18.0 Maximum Volume (uL) 32.2 ± 2.3 36.4 ± 3.4 36.9± 1.1 40.8 ± 1.6 38.1 ± 1.5 48.9 ± 4.4 Minimum Volume (uL) 13.7 ± 2.415.8 ± 3.3 14.7 ± 1.9 25.7 ± 0.9 18.4 ± 0.5 36.5 ± 3.7 End-systolicVolume (uL) 14.7 ± 2.8 16.9 ± 3.3 15.5 ± 2.1 28.0 ± 0.7 19.3 ± 0.5 40.2± 4.3 End-diastolic Volume (uL) 30.6 ± 2.4 34.5 ± 3.2 35.2 ± 1.1 39.8 ±1.6 36.8 ± 1.4 47.2 ± 4.1 Maximum Pressure (mmHg) 93.1 ± 3.5 149.2 ±4.8  92.6 ± 0.8 153.5 ± 6.1  93.6 ± 5.0 169.8 ± 10.2 Minimum Pressure(mmHg)  4.9 ± 1.3  3.2 ± 0.4  3.6 ± 0.1  7.3 ± 3.6  4.1 ± 0.5  6.2 ± 1.9End-systolic Pressure (mmHg) 87.3 ± 4.3 139.4 ± 2.8  89.2 ± 1.0 149.6 ±5.0  90.5 ± 4.9 168.3 ± 9.8  End-diastolic Pressure (mmHg) 14.0 ± 3.210.6 ± 2.7 13.0 ± 0.7 16.8 ± 4.8 16.5 ± 1.4 16.9 ± 3.1 Stroke Volume(uL) 18.6 ± 1.0 20.6 ± 0.7 22.2 ± 2.3 15.1 ± 1.2 19.7 ± 1.4 12.4 ± 1.0Ejection Fraction (%) 58.7 ± 5.1 57.9 ± 4.5 60.0 ± 5.3 36.8 ± 1.9 51.5 ±1.6 25.8 ± 2.0 Cardiac Output (uL/min) 5113.5 ± 819.2 5879.1 ± 714.06114.8 ± 897.0 4108.6 ± 310.3 5066.0 ± 653.3 3893.8 ± 466.1 Stroke Work(mmHg * uL) 1339.6 ± 134.0 2196.3 ± 94.6  1577.8 ± 134.4 1477.8 ± 99.6 1451.8 ± 130.4 1179.2 ± 104.1 Arterial Elastance (Ea) (mmHg/uL)  4.8 ±0.4  6.8 ± 0.3  4.1 ± 0.4 10.1 ± 0.7  4.7 ± 0.4 14.1 ± 1.7 dPdt max(mmHg/sec) 5481.6 ± 491.1 6785.3 ± 434.2 6036.0 ± 352.9 5133.2 ± 621.45755.8 ± 652.9 6454.4 ± 712.0 dPdt min (mmHg/sec) −5049.6 ± 426.9 −7427.5 ± 685.3  −4743.3 ± 287.7  −5484.75 ± 412.2  −4564.5 ± 525.8  −7625 ± 586.5 dVdt max (uL/sec) 883.0 ± 61.2 758.0 ± 29.8 856.5 ± 27.41152.8 ± 206.3 1188.0 ± 114.1 1041.2 ± 109.6 dVdt min (uL/sec) −679.6 ±71.4  −696.3 ± 30.6  −703.5 ± 52.2  −921.0 ± 158.0 −1000.5 ± 76.8 −938.4 ± 126.2 P@dVdt max (mmHg)  9.0 ± 2.5  7.4 ± 2.6  4.6 ± 0.4 10.3 ±3.4  6.2 ± 1.0 13.3 ± 4.5 P@dPdt max (mmHg) 44.1 ± 2.1 46.3 ± 3.5 49.0 ±2.6 47.1 ± 2.8 49.6 ± 5.6 52.8 ± 3.6 V@dPdt max (uL) 31.2 ± 2.4 35.5 ±3.5 35.0 ± 1.1 39.7 ± 1.6 37.0 ± 1.5 47.3 ± 4.4 V@dPdt min (uL) 14.7 ±2.6 17.1 ± 3.2 15.6 ± 1.9 27.0 ± 0.7 19.2 ± 0.4 39.0 ± 4.3 Tau_w (msec)11.4 ± 1.2  8.6 ± 0.7 10.7 ± 0.9 11.2 ± 1.3 11.3 ± 0.5  8.8 ± 0.4 Tau_g(msec) 15.8 ± 1.5 12.1 ± 1.2 17.5 ± 0.7 17.4 ± 1.0 17.5 ± 1.0 15.6 ± 1.0Maximal Power (mWatts)  6.4 ± 0.6  9.5 ± 0.4  6.8 ± 0.5  8.8 ± 0.5  7.3± 0.7  9.0 ± 0.5 Preload adjusted maximal power  74.8 ± 16.5  85.0 ±12.9 55.5 ± 2.4 57.3 ± 7.4 53.6 ± 3.0  46.1 ± 11.5 (mWatts/

N5L{circumflex over ( )}2)

In addition to functional parameters histology by Hematoxylin/Eosin (HE)staining is performed on cardiac tissue from AB mice and control mice at2, 4, and 8 weeks. Histology confirms the expected necrotic andremodeling processes for the AB mice, whereas heart tissue in shamoperated mice does not show any significant changes. At two weeks aftersurgery the ventrical of a ligated mouse shows significant leftventricular hypertrophy which after four weeks has further progressedand at eight weeks post surgery closely resembles endstage dilatedcardiomyopathy.

Example 2 Sample Preparation and Mass Spectroscopy

Heart Homogenization and Organelle Isolation:

Hearts are isolated, atria removed, the ventricles carefully minced witha razor blade and rinsed extensively with ice-cold PBS (phosphatebuffered saline) to remove excess blood. Tissue is homogenized for 30 susing a loose fitting hand-held glass homogenizer in 10 ml lysis buffer(250 mM sucrose, 50 mM Tris-HCl pH 7.6, 1 mM MgCl2, 1 mM DDT(dithiothreitol), and 1 mM PMSF (phenylmethylsulphonyl fluoride). Allsubsequent steps are performed at 4° C. The lysate is centrifuged in abenchtop centrifuge at 800×g for 15 min; the supernatant serves as asource for cytosol, mitochondria, and microsomal fractions. The pelletcontaining nuclei is diluted in 8 ml of lysis buffer and layered onto 4ml of 0.9 M sucrose buffer (0.9 M sucrose, 50 nM Tris-HCl pH 7.6, 1 mMMgCl2, 1 mM DDT, and 1 mM PMSF) and centrifuged at 1000×g for 20 min at4° C. The resulting pellet is resuspended in 8 ml of a 2 M sucrosebuffer (2 M sucrose, 50 mM Tris-HCl pH 7.4, 5 mM MgCl2, 1 mM DTT, and 1mM PMSF), layered onto 4 ml of 2 M sucrose buffer and pelleted byultracentrifugation at 150,000×g for 1 h (Beckman SW40.1 rotor). Thenuclei are recovered as a pellet. The mitochondria are isolated from thesupernatant by re-centrifugation at 7500×g for 20 min at 4° C.; theresulting pellet is washed twice in lysis buffer. Microsomes arepelleted by ultracentrifugation of the post-mitochondrial cytoplasm at100,000×g for 1 h in a Beckman SW41 rotor. The supernatant served as thecytosolic fraction (=cyto).

Organelle Extraction:

Soluble mitochondrial proteins are extracted by incubating themitochondria in hypotonic lysis buffer (10 mM HEPES, pH 7.9, 1 mM DTT, 1mM PMSF), for 30 min on ice. The suspension is sonicated briefly anddebris removed by centrifugation at 13,000×g for 30 min. The supernatantserves as the “mito 1” fraction. The resulting insoluble pellet isresuspended in membrane detergent extraction buffer (20 mM Tris-HCl, pH7.8, 0.4 M NaCl, 15% glycerol, 1 mM DTT, 1 mM PMSF, 1.5% Triton-X-100)and shaken gently for 30 min followed by centrifugation at 13,000×g for30 min; the supernatant served as “mito 2” fraction.

Membrane-associated proteins are extracted by resuspending themicrosomes in membrane detergent extraction buffer. The suspension isincubated with gentle shaking for 1 h and insoluble debris removed bycentrifugation at 13,000×g for 30 min. The supernatant serves as the“micro” fraction.

Digestion of Organelle Extracts and MudPITAnalysis:

An aliquot of about 100 μg total protein (as determined by Bradfordassay) from each fraction is precipitated overnight with 5 vol ofice-cold acetone at about 20° C., followed by centrifugation at 13,000×gfor 15 min. The protein pellet is solubilized in a small volume of 8 Murea, 50 mM Tris-HCl, pH 8.5, 1 mM DTT, for 1 h at 37° C., followed bycarboxyamidomethylation with 5 mM iodoacetamide for 1 h at 37° C. in thedark. The samples are then diluted to 4 M urea with an equal vol of 100mM ammonium bicarbonate, pH 8.5, and digested with a 1:150-fold ratio ofendoproteinase Lys-C (Roche Diagnostics, Laval, Quebec, Canada) at 37°C. overnight. The next day, the samples are diluted to 2 M urea with anequal vol of 50 mM ammonium bicarbonate pH 8.5, supplemented with CaCl2to a final concentration of 1 mM, and incubated overnight with Poroszymetrypsin beads (Applied Biosystems, Streetsville, Ontario, Canada) at 30°C. with rotation. The resulting peptide mixtures are solidphase-extracted with SPEC-Plus PT C18 cartridges (Ansys Diagnostics,Lake Forest, Calif.) according to the instructions of the manufacturerand stored at −80° C. until further use. A fully-automated 20 h long12-step multi-cycle MudPIT procedure is set up as described (Mol. CellProteom. 2 (2003) 96-106). Briefly, an HPLC quaternary pump isinterfaced with an LCQ DECA XP ion trap mass spectrometer (ThermoFinnigan, San Jose, Calif.). A 100-μm i.d. fused silica capillarymicrocolumn (Polymicro Technologies, Phoenix, Ariz.) is pulled to a finetip using a P-2000 laser puller (Sutter Instruments, Novato, Calif.) andpacked with 8 cm of 5 μm Zorbax Eclipse XDB-C18 resin (AgilentTechnologies, Mississauga, Ontario, Canada), followed by 6 cm of 5 μmPartisphere strong cation exchange resin (Whatman, Clifton, N.J.).Individual samples are loaded manually onto separate columns using apressure vessel. Chromatography solvent conditions are exactly asdescribed in Kislinger, T., et al., Mol. Cell Proteom. 2 (2003) 96-106.

Protein Identification and Validation:

The SEQUEST database search algorithm is used to match peptide tandemmass spectra to peptide sequences in a locally-maintained minimallyredundant FASTA formatted database populated with mouse and humanprotein sequences obtained from the Swiss-Prot/TrEMBL and IPI databases.To statistically assess the empirical False-Discovery Rate to controlfor, and hence, minimize false positive identifications, all of thespectra are searched against protein sequences in both the normal(Forward) and inverted (Reverse) amino acid orientations (Kislinger, T.,et al., Mol. Cell Proteom. 2 (2003) 96-106). The STATQUEST filteringalgorithm is then applied to all putative search results to obtain ameasure of the statistical reliability (confidence score) for eachcandidate identification (cutoff p-value ≦15, corresponding to an 85% orgreater likelihood of being a correct match). High-confidence matchesare parsed into an in-house SQL-type database using a Perl-based script.The database is designed to accommodate database search results andspectral information (scan headers) for multiple peptides matching to agiven protein, together with information regarding the sample name,experiment number, MudPIT step, organelle source, amino acid sequence,molecular mass, isoelectric point, charge, and confidence level. Onlythose proteins with a predicted confidence p value of 95% or more, andfor which at least two spectra are collectively detected, are retainedfor further analysis.

Example 3 Statistical Evaluation of the Data Obtained in the ModelSystems

3.1: Statistical Methods Used to Generate p-Values of DifferentialExpression for the R9C Mouse Model

The raw data obtained with the methods as described in Example 2consists of 6190 proteins each with spectral counts, the sum of allspectra associated with the protein, for each of the 137 differentexperimental runs. The raw data, 6190 subset of proteins, is subjectedto global normalization which first separates the data within each runinto an equal number of groups, set at 100 for our analysis, based ontheir spectral counts. LOESS (Cleveland, W. S. and Devlin, S. J.,Journal of the American Statistical Association 83 (1988) 596-610) isthen performed on each group (1-100) adjusting for differences inspectral counts across a set of genes with similar spectral counts.

Based on our raw data we constructed two linear models, the first modeluses control/disease, time (8W, 16W, end) and location (cyto, micro,mitoI, mitoII) as factors and is described using:run count=β0+β1time+β2time²+β3location+β4control  (1)The second model uses only time (8W, 16W, end) and location (cyto,micro, mitoI, mitoII) as factors and is described using:run count=β0+β1time+β2time²+β3location  (2)where β0 is the intercept term and β1, β2, β3, and β4 are the slopeestimates for the variables time, time squared, location, andcontrol/disease.

The two models are compared using Anova, with the null hypothesis beingthat there is no difference between the two models. A low p-value thenindicates that there is not enough proof to say the two models are thesame. The extra information indicates the state (i.e., control/disease)appears to be a significant component of the model. In order to extractproteins that have a significant change in relative protein abundancebetween our control and disease models our list of 6190 proteins isranked based on their computed p-values. This generates a set of 593proteins with p-values <0.05.

In order to account for multiple hypothesis testing from the above modelthe p-values are then corrected using false discovery rate (FDR)correction, specifically Benjamini-Hochberg FDR correction (Benjamini,Y., and Hochberg, Y., Journal of the Royal Statistical Society B. 57(1995) 289-300). This generates a set of 40 proteins with correctedp-values <0.05 for the R9C mouse model.

3.2: Statistical Methods Used to Generate p-Values of DifferentialExpression for the Aortic Banding Mouse Model

The same data analysis is applied to the datasets for the aortic bandingmouse model as described above for the R9C mouse model.

Example 4 Detection of the Marker SLIM-1 by Western Blot Assay

Crude tissue lysates are obtained from R9C mouse heart tissue samples.In brief, heart tissue is minced, ground up in a dounce homogenizer andsubjected to a centrifuge spin of 8,000 g for 30 min to remove nucleiand cell debris. The supernatant is used for Western Blotting.

SDS-PAGE and Western-Blotting are carried out using reagents andequipment of Invitrogen, Karlsruhe, Germany. For each tissue sampletested, 10 μg of the cytosolic fraction are diluted in reducing NuPAGE®(Invitrogen) SDS sample buffer and heated for 10 min at 95° C. Samplesare run on 4-12% NuPAGE® gels (Tris-Glycine) in the MES running buffersystem. The gel-separated protein mixture is blotted onto nitrocellulosemembranes using the Invitrogen XCell II™ Blot Module (Invitrogen) andthe NuPAGE® transfer buffer system. The membranes are washed 3 times inPBS/0.05% TWEEN®-20 (a polysorbate 20, ICI Americas Inc.) and blockedwith Roti-Block blocking buffer (A151.1; Carl Roth GmbH, Karlsruhe,Germany) for 2 h. The primary antibody, rabbit polyclonal to four andhalf LIM domain (FHL-1) (IMG-3374; Imgenex/Cedarlane) is diluted inRoti-Block blocking buffer and incubated with the membrane for 1 h. Themembranes are washed 6 times in PBS/0.05% TWEEN®-20. The specificallybound primary SLIM-1 antibody is labeled with a POD-conjugatedpolyclonal anti-rabbit IgG antibody, diluted to 10 mU/ml in 0.5 xRoti-Block blocking buffer. After incubation for 1 h, the membranes arewashed 6 times in PBS/0.05% TWEEN®-20. For detection of the boundPOD-conjugated anti-rabbit antibody, the membrane is incubated with theLumi-Light^(PLUS) Western Blotting Substrate (Order-No. 2015196, RocheDiagnostics GmbH, Mannheim, Germany) and exposed to an autoradiographicfilm.

Results of a typical experiment are shown in FIG. 3. A strongoverexpression of SLIM-1 is observed in tissue samples derived R9Cexperimental animals suffering from heart failure versus tissue samplesderived at corresponding time points from a healthy mouse.

Example 5 ELISA for the Measurement of SLIM-1 in Human Serum and PlasmaSamples

For detection of SLIM-1 in human serum or plasma, a sandwich ELISA isdeveloped. For capturing of the antigen, aliquots of an anti-SLIM-1polyclonal antibody obtained by immunization of rabbits with SLIM-1produced in HEK cells and for detection of the antigen a serum producedin goats with a SLIM fragment consisting of amino acids 233-246,respectively, are used and conjugated with biotin and digoxygenin,respectively.

Streptavidin-coated 96-well microtiter plates are incubated with 100 μlbiotinylated anti-SLIM-1 polyclonal antibody for 60 min at 10 μg/ml in1x PBS solution. After incubation, plates are washed three times with 1xPBS+0.02% TWEEN®-20 (a polysorbate 20), blocked with PBS+1% BSA (bovineserum albumen) and then washed again three times with 1x PBS+0.02%TWEEN®-20. Wells are then incubated for 1 h with either a serialdilution of the recombinant SLIM-1 as standard antigen or with dilutedserum or plasma samples (1:5) from patients or control individuals,respectively. After binding of SLIM-1, plates are washed three timeswith 1x PBS+0.02 TWEEN®-20. For specific detection of bound SLIM-1,wells are incubated with 100 μl of digoxigenylated anti-SLIM-1polyclonal antibody for 45 min at 0.5 μg/ml in 1x PBS, 1% BSA.Thereafter, plates are washed three times to remove unbound antibody. Ina next step, wells are incubated with 75 mU/ml anti-digoxigenin-PODconjugates (Roche Diagnostics GmbH, Mannheim, Germany, Catalogue No.1633716) for 30 min in 1x PBS, 1% BSA. Plates are subsequently washedsix times with the same buffer. For detection of antigen-antibodycomplexes, wells are incubated with 100 μl ABTS solution (RocheDiagnostics GmbH, Mannheim, Germany, Catalogue No. 11685767) and theoptical density (OD) is measured after 30 min at 405 and 492 nm with anELISA reader.

10 serum samples obtained from different patients with heart failure (HFsamples) and 10 sera of normal healthy donors (NHS) are tested. Afterperforming the assay procedure as described above the following results(see Table 3) are obtained with these samples:

TABLE 3 SLIM-1 ELISA results (assay development samples) OD HF Samples5078 0.360 5084 0.553 5085 0.353 5100 0.442 5001 0.318 5104 0.475 51070.344 5112 0.361 5113 0.222 5114 0.427 MW 0.385 NHS Samples   2 0.232 33 0.465  36 0.299  41 0.305  44 0.349  51 0.164  57 0.336  60 0.206 62 0.117  77 0.163 MW 0.263

The data summarized in Table 3 are also represented in FIGS. 4 and 5. Asobvious from FIGS. 4 and 5 the SLIM-1 levels are in average higher inthe sera obtained from patients with HF as compared to the levels foundin the samples obtained from control individuals.

Example 6 Marker Combinations Comprising the Marker SLIM-1 in theAssessment of Heart Failure

6.1: The Marker Combination NT-proBNP and SLIM-1

The marker combination NT-proBNP and SLIM-1 is evaluated for thedifferentiation of patients in stage B and stages C plus D,respectively. Diagnostic accuracy is assessed by analyzing individualliquid samples obtained from well-characterized groups of individuals,i.e., 50 individuals in stage B according to the ACA/ACC criteria forclassification of HF and 50 patients suffering from HF and having stageC according to the ACA/ACC criteria for classification of HF. NT-proBNPas measured by a commercially available assay (Roche Diagnostics,NT-proBNP-assay (Cat. No. 03 121 640 160 for ELECSYS Systems immunoassayanalyzer) and SLIM-1 measured as described above are quantified in aserum sample obtained from each of these individuals. ROC-analysis isperformed according to Zweig, M. H., and Campbell, supra. Discriminatorypower for differentiating patients in stage C from individuals in stageB for the combination of SLIM-1 with the established marker NT-proBNP iscalculated by regularized discriminant analysis (Friedman, J. H., J. ofthe American Statistical Association 84 (1989) 165-175).

6.2: The Marker Combination Troponin T and SLIM-1

The marker combination troponin T and SLIM-1 is evaluated for thedifferentiation of patients suffering from an acute cardiac event frompatients suffering from chronic heart disease, respectively. Diagnosticaccuracy is assessed by analyzing individual liquid samples obtainedfrom well-characterized groups of individuals, i.e., 50 individualsdiagnosed as having an acute cardiac event and 50 individuals diagnosedas having chronic cardiac disease. Troponin T as measured by acommercially available assay (Roche Diagnostics, troponin T-assay (Cat.No. 201 76 44 for ELECSYS Systems immunoassay analyzer, RocheDiagnostics GmbH) and SLIM-1 measured as described above are quantifiedin a serum sample obtained from each of these individuals. ROC-analysisis performed according to Zweig, M. H., and Campbell, G., supra.Discriminatory power for differentiating patients in stage C fromindividuals in stage B for the combination of SLIM-1 with theestablished marker troponin T is calculated by regularized discriminantanalysis (Friedman, J. H., J. of the American Statistical Association 84(1989) 165-175).

6.3: The Marker Combination CRP and SLIM-1

The marker combination C-reactive protein and SLIM-1 is evaluated forthe differentiation of patients diagnosed as having a cardiomyopathyversus controls not suffering from any confounding heart disease,respectively. Diagnostic accuracy is assessed by analyzing individualliquid samples obtained from well-characterized groups of 50 individualswith cardiomyopathy and of 50 healthy control individuals. CRP asmeasured by a commercially available assay (Roche Diagnostics, CRP-assay(TINA-QUANT C-reactive protein (latex) high sensitive assay—RocheDiagnostics GmbH Cat. No. 11972855 216) and SLIM-1 measured as describedabove are quantified in a serum sample obtained from each of theseindividuals. ROC-analysis is performed according to Zweig, M. H., andCampbell, G., supra. Discriminatory power for differentiating patientsin stage C from individuals in stage B for the combination of SLIM-1with the established marker CRP is calculated by regularizeddiscriminant analysis (Friedman, J. H., J. of the American StatisticalAssociation 84 (1989) 165-175).

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be appreciated by oneskilled in the art, from a reading of the disclosure that variouschanges in form and detail can be made without departing from the truescope of the invention in the appended claims.

What is claimed is:
 1. A method of detecting an amount of four and a half LIM domain-1 (SLIM-1) in a subject, the method comprising: contacting a portion of a sample from the subject with a detectable antibody having specific binding affinity for SLIM-1 and wherein the antibody binds within amino acids 233-246 of SEQ ID NO:1, thereby forming a detectable complex between the detectable antibody and any SLIM-1 present in the sample, wherein the sample is selected from the group consisting of serum, plasma and blood; separating the detectable complex from uncomplexed antibody; and quantifying a signal from the detectable complex, the signal being proportional to an amount of SLIM-1 in the sample, thereby detecting an amount of SLIM-1 in the sample.
 2. The method of claim 1, further comprising determining an amount of at least one additional marker in a portion of the sample from the subject.
 3. The method of claim 2, wherein the additional marker is selected from the group consisting of troponin T, NT-proBNP, and CRP. 